| The human genome is the whole set of DNA arrayed in 24 distinct chromosomes,which consists of 3.2 billions of base pairs (bp). The genomic DNA is 99.9% identical tothat of other humans and 0.1% is variable. One of the fruits of the Human GenomeProject is the discovery of millions of DNA sequence variants in the human genome. Theuse of these genetic markers has been playing an increasing part in genetics studies. Mostvariation of the human genome is attributable to single nucleotide polymorphisms (SNPs),which contributes to the differences between individuals in physical appearances,susceptibility to a disease and response to medication with drugs. SNPs have been widelyused. A dense set of SNP markers opens up the possibility of studying the genetic basis ofcomplex disease. In recent years, SNPs have been used as new DNA markers to replacemicrosatellites in mapping of disease-related genes in humans. There is no doubt thatSNPs will play a key role in identifying and cloning of disease-related genes,investigating the mechanism of the genome-environment interaction and gene-geneinteraction.Schizophrenia is a serious mental disorder with a lifetime prevalence rate of 1% inthe general population worldwide. It was characterized by the abnormal mental functionsand disturbed behaviors, which characteristically appear as a series of clinical features,such as positive and negative symptoms, and disturbances in basic cognitive functions.Because the illness causes heavy economical and social burdens to families and societies,it is very important to establish a procedure of treating and preventing schizophrenia.While the cause for schizophrenia remains unknown, several lines of evidence fromfamily, twin and adoption suggest that genetic factors are likely to play an essential role inthe developing of schizophrenia and influence susceptibility to schizophrenia.Epidemiological data have demonstrated that schizophrenia is not a simple Mendeliandisease but looks like a complex disease involving several genes with each susceptiblitygene having only a modest individual effect. Genome-wide scan has suggested that thefollowing chromosomal regions may contain several susceptibility genes contributing toschizophrenia, including 1q21-22, 5q31-33, 6p21-24, 6q25-26, 8p21-22, 10p11-15,13q14-33 and 22q11-13. To validate these initial findings, in the present study we havefocused on identification of the candidate susceptibility genes on the 5q31-35 regionusing a family-based LD.The family trio consists of healthy fathers, healthy mothers and affected offspringwith schizophrenia. By using bioinformatics methods 7 SNPs were chosen on the 5q31-35region including rs888957 present in the 5-HTR4 locus,rs33409 in the SYNPO locus,rs2071220 in the GLRA1 locus,rs1428157 in the X locus,rs154110 in the Y locus,rs2066949 in the GABRB2 locus and rs211014 in the GABRG2 locus. Meanwhile,4 SNPs were chosen in the KPNB locus including rs11871606 present in the KPNB1locus on the 17q21 region,rs34667 and rs266443 in the KPNB2 locus on 5q13 region aswell as rs2588014 in the KPNB3 locus on 13q32 region.SNPs were genotyped using PCR-based RFLP analysis. Genotyping data were putinto the SPSS database. The Hardy-Weinberg (H-W) equilibrium was tested for genotypefrequency distributions of SNPs using the goodness of fit test. The LD between pairedSNPs was estimated with UNPHASED programs. The haplotype-based haplotype relativerisk (HHRR) test and the transmission disequilibrium test (TDT) were applied to detectallelic association between SNPs and schizophrenia. To elucidate genetic heterogeneity, inaddition, schizophrenic patients were sub-grouped based on their clinical symptoms andthe genetic association between SNPs and clinical subgroups was then analyzed.Combined effects of paired SNPs were tested by UNPHASED programs .The patientswere divided into two groups according to the clinical psychotic symptoms. We analyzedpsychotic symptoms versus allelic and genotypic frequency for each SNP.The details of methodology and major results obtained in this study is as follows:1. The H-W equilibriumThe goodness of fit test showed that genotype frequency distributions of all subjectswere not deviated from the H-W equilibrium, thus these samples were suitable for thegenetic analysis.2. LD between paired SNPsThe estimated LD showed that rs2071220 and rs1428157,rs34667 and rs266443were in the same LD block with UNPHASED programs.3. Association between SNPs and schizophrenia3.1 The TDT analysis :The TDT analysis showed that rs2588014 was associated with schizophrenia(P=0.019). The rs2588014 present in the KPNB3 is a C to T base change andheterozygous parents have excessively transmitted allele C to their affected offspring,suggesting that the haplotype containing rs2588014(T)may carry disease-resistant variantfor schizophrenia.3.2 The HHRR analysis:The HHRR analysis revealed an allelic association between rs2588014 andschizophrenia. This finding was consistent with TDT result.3.3 Analysis for clinical subgroups:The results showed that rs2071220, rs1428157, rs154110, rs2066949, rs211014,rs11871606 and rs34667 were associated with schizophrenia. Since the time at whichSNPs occurred differs, each SNP may have its own genetic heritage with a differenthaplotype and non-random association. They may affect each other in allele frequencydistribution, so as to reduce the power to detect their association with the illness. Theadvantage of clinical subgroup analysis is to limit the interference from different LDsignals to reduce the false negative results and validate the hypothesis of geneticheterogeneity.3.4 Analysis for combined effects of multiple locus:3.4.1 Analysis for haplotype transmissionUNPHASED analysis showed that the rs2071220-rs1428157 and rs34667-rs266443haplotype systems did not associated with schizophrenia (P>0.05).3.4.2 Analysis for conditional test:The conditional test was used to test the combined effect of distint loci on the diseaseby conditioning on allele(COA)or by conditioning on genotype(COG).The COA testshowed a disease association for the rs888957-rs2588014 and rs2066949-rs2588014combination. The COG test also showed an association for the above locus combinations.4. The association between SNPs and psychotic symptoms of schizophrenia4.1 The association between rs888957 and psychotic symptoms of schizophreniaThe distribution of allele and genotype frequencies was significantly differentbetween the patients with delusion of love and those without the symptom (P=0.000,P=0.001 respectively). It suggested that the rs888957 was associated with delusion oflove,one of positive symptoms of schizophrenia.4.2 The association between rs33409 and psychotic symptoms of schizophreniaThe distribution of allele and genotype frequencies was significantly differentbetween the patients with apathy and those without the symptom (P=0.021,P=0.025respectively). It suggested that the rs33409 was associated with the severity of apathy,one of negative symptoms of schizophrenia.4.3 The association between rs2071220 and psychotic symptoms of schizophreniaThe distribution of allele and genotype frequencies was significantly differentbetween the patients with delusion of jealousy as well as incoherentce of thinking andthose without the symptoms (P=0.028,P=0.033;P=0.021,P=0.019 respectively). Itsuggested that the rs2071220 was associated with delusion of jealousy and incoherentce ofthinking of positive symptoms of schizophrenia. The distribution of genotype frequencieswas significantly different between the patients with poverty of thought and premorbidpersonality and those without the symptoms (P=0.031,P=0.012 respectively). Itsuggested that the rs2071220 was associated with poverty of thought and premorbidpersonality of negative symptoms of schizophrenia.4.4 The association between rs1428157 and psychotic symptoms of schizophreniaThe distribution of allele frequencies was significantly different between the patientswith delusion of being revealed and those without the symptom (P=0.034). It suggestedthat the rs1428157 was associated with delusion of being revealed,one of positivesymptoms of schizophrenia. The distribution of allele and genotype frequencies wassignificantly different between the patients with incoherentce of thinking and thosewithout the symptom (P=0.015,P=0.024 respectively). It suggested that the rs1428157was associated with incoherentce of thinking,one of positive symptoms of schizophrenia.4.5 The association between rs154110 and psychotic symptoms of schizophreniaThe distribution of allele frequencies was significantly different between the patientswith other delusion and those without the symptom (P=0.041). It suggested that thers154110 was associated with other delusion,one of positive symptoms of schizophrenia.The distribution of genotype frequencies was significantly different between the patientswith delusion of persecution and those without the symptom (P=0.013). It suggested thatthe rs154110 was associated with delusion of persecution,one of positive symptoms ofschizophrenia.4.6 The association between rs2066949 and psychotic symptoms of schizophreniaThe distribution of allele and genotype frequencies was significantly differentbetween the patients with genuine auditory hallucination as well as hallucination-delusionsyndrome and those without the symptoms (P=0.003,P=0.017;P=0.008,P=0.025respectively). It suggested that the rs2066949 was associated with genuine auditoryhallucination and hallucination-delusion syndrome of positive symptoms ofschizophrenia.4.7 The association between rs34667 and psychotic symptoms of schizophreniaThe distribution of allele and genotype frequencies was significantly differentbetween the patients with hallucination-delusion syndrome and those without thesymptoms (P=0.039 ,P=0.049 respectively). It suggested that the rs2066949 wasassociated with hallucination-delusion syndrome, one of positive symptoms ofschizophrenia.4.8 The association between rs266443 and psychotic symptoms of schizophreniaThe distribution of allele and genotype frequencies was significantly differentbetween the patients with delusion of persecution and those without the symptom(P=0.019,P=0.012 respectively). It suggested that the rs266443 was associated withdelusion of persecution, one of positive symptoms of schizophrenia.4.9 The association between rs2588014and psychotic symptoms of schizophreniaThe distribution of genotype frequencies was significantly different between thepatients with delusion of jealiusy as well as apathy and those without the symptoms(P=0.03, P=0.036 respectively). It suggested that the rs2588014 was associated withdelusion of jealiusy, one of positive symptoms of schizophrenia, and the severity ofapathy, one of negative of schizophrenia.Taken together, the present study demonstrated that KPNB3 might play an importantrole in the developing of schizophrenia. Eight SNPs, including rs888957, rs2071220,rs1428157, rs154110, rs2066949, rs34667, rs266443 and rs2588014, were associated withsome positive symptoms of schizophrenia. The rs33409,rs2071220 and rs2588014 werefound to be associated with poverty of thought,apathy and premorbid personality thatwere classified as three negative symptoms of schizophrenia. These findings are veryimportant for elucidating the molecular genetic mechanisms of schizophrenia, and also forgenetic diagnosis, developing new drugs and prediction of schizophrenia.
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